The incidence of melanoma has been on the rise over the last 30 years with the 5 year survival rate approaching 2% for patients with metastatic melanoma; veterans are up to 4.5 times more likely to develop melanoma than the general population. These dismal statistics are partly due to the fact that melanoma is notoriously resistant to cancer therapies. We reported that melanoma cell lines and patient samples express elevated levels of the IL-1 receptor-associated kinase-4 (IRAK-4) and IRAK-1. IRAK-4 plays a central role in Toll-like receptor, IL-1, IL-18 and IL-33 signaling. Attenuating IRAK-1,-4 signaling in melanoma cells with pharmacological inhibitors or siRNA diminished the production of various chemokines/cytokines known to promote angiogenesis and the induction of tumor-associated fibroblasts. IRAK inhibition also enhanced the effects of certain chemotherapies in vitro and in tumor-bearing mice. In contrast, the activation of IRAK-4 signaling in melanoma cells increased the production of these cytokines/chemokines and promoted chemotherapeutic resistance. Supernatant from tumor cells engineered to overexpress the IRAK-4 gene augmented endothelial cell proliferation and activated human fibroblasts, as demonstrated by increased expression of Tenascin-C and -SMA. Interestingly, while IRAK-4 is known to localize in the cytosol, we observed that in melanoma IRAK-4 translocates to the nucleus and this is increased upon chemotherapy exposure. The overarching hypothesis is that IRAK-4 nuclear mobilization promotes chemoresistance and that IRAK signaling orchestrates changes in the tumor microenvironment that promotes tumor growth and metastases. The proposed studies are focused on gaining greater mechanistic insights (molecular and cellular) on how IRAK-4 signaling in tumor cells: (1) influences angiogenesis, chemoresistance and metastases, (2) changes the tumor microenvironment, and (3) can be used as a therapeutic target to enhance the efficacy of chemotherapies. Aim 1 is to gain a mechanistic understanding regarding the role that nuclear IRAK-4 plays in chemotherapy resistance and gene expression. Determining the function of nuclear IRAK-4 is novel and independent of known mechanisms of IRAK-4-mediated activation. This will be investigated using melanoma lines engineered to express IRAK-4 variants including wild type, shRNA-IRAKs, kinase dead IRAKs, and IRAK- 4 SUMO, in which SUMOylation sites have been mutated to increase cytosolic levels but prevent nuclear mobilization. Aim 2 is to determine the effect that inhibiting IRAK-4 in melanoma has on metastasis and angiogenesis. We will use an inducible model of melanoma in mice (BRAFV600PTEN mice) as well as human and mouse melanoma cells engineered to overexpress or knockdown IRAK-4 to monitor tumor neovascularization and metastases using in vivo and ex vivo models. Aim 3 is to discover the role that IRAK-4 signaling in melanoma plays in altering the tumor microenvironment to promote tumor growth. Building on preliminary data showing that IRAK-4 signaling in melanoma induces the expression of factors that support tumor associated fibroblasts, this aim will explore the cellular mechanism and investigate the previously unrecognized concept that IRAK signaling within tumor cells is critical for orchestrating the generation of a microenvironment favoring tumor growth and metastasis. Aim 4 seeks to determine the therapeutic efficacy of targeted delivery of IRAK inhibitor to tumors when combined with chemotherapies. We will use anti-PD-L1 antibodies to deliver FDA-approved chemotherapies plus IRAK-1,-4 inhibitor (currently in Phase II/III clinical trials). The results from these studies will provide a greater molecular and cellular understanding of how IRAK-4 signaling in melanoma enhances chemoresistance and metastasis and will identify new therapeutic targets to enhance chemotherapeutic responses.